Compositions containing a peptide and polylactic-glycolic acid suitable for preparing subcutaneous implants with an extended release period

ABSTRACT

Compositions comprising a peptide and polylactic-glycolic acid (PLGA) wherein the distribution of the size of the particles of the peptide in the PLGA results highly heterogeneous, suitable to the preparation of subcutaneous implants having a release time at least equal to 6 months.

This application is a 371 of PCT/EP 99/09536 P/ED Jun. 12, 1999.

1. Field of the Invention

The present invention refers to compositions comprising a peptide andpolylactic-glycolic acid suitable for the preparation of subcutaneousimplants.

2. Prior Art

Compositions made up of mixtures of a drug with a polymer of lactic acidor a polymer of glycolic acid or with a copolymer of lactic acid andglycolic acid, as described in the U.S. Pat. No. 3,773,919 (Du Pont) arewell known.

These compositions are indicated for parenteral administration and havethe characteristics of releasing effective quantities of the drug over aset period of time.

The drug and the polymeric substance can be combined in accordance withany of the known techniques or the particles of the drug can be coatedin the polymer operating in accordance with known techniques.

The U.S. Pat. No. 4,767,628 (ICI) describes compositions containing apeptide and a polymer of lactic acid or a copolymer of lactic acid andglycolic acid.

When preparing the compositions, the peptide and the (co)polymer aredissolved in a solvent which can be the same or different for the twosubstances, for Example dioxane or water, and then the two solutions aremixed.

The subsequent operations consist of removing the solvent at lowtemperature and in extruding the powder obtained in this way.

In this way a composition in the form of cylinders is obtained in whichthe peptide is distributed homogeneously throughout the polymer.

It is already known from the U.S. Pat. No. 5,366,734 (Zeneca) that thecompositions covered by the U.S. Pat. No. 4,767,628 referred to abovecan be used in preparing subcutaneous implants.

The polymer of lactic acid and the copolymer of lactic acid and glycolicacid are incompatible with the peptide therefore diffusion of thepeptide through the polymer is impossible.

When these implants are introduced into a buffer solution at 37° C., thewater penetrates and diffuses in the implant and is distributed betweenthe polymer and the peptide forming regions of hydrated peptides.

The first stage in releasing the peptide described in the U.S. Pat. No.5,366,734 is a stage of diffusion caused by the polymer swelling.

When the polymer swells this allows channels of hydrated peptide to formwhere the peptide diffuses to the surface.

If swelling stops, the peptide is no longer released.

The second stage of release is caused by the polymer matrix degrading.

During this stage holes and cracks form in the matrix which allow therelease of the hydrated peptides which are still isolated in the matrix.

The total release time is limited to the sum of the release times foreach stage. However the maximum release time observed is in the order ofthree months.

In the application for international patent WO 98/09613 (Deghenghi) aprocess for preparing subcutaneous implants capable of releasingbioactive peptides is described.

This process consists of the following stages:

milling a copolymer of lactic acid and glycolic acid,

wetting the copolymer with an aqueous slurry of a peptide (in theExamples an aqueous solution of avoreline acetate is used):

mixing this copolymer with the aforementioned slurry so as to obtain ahomogeneous mixture;

drying this mixture at a temperature of no higher than 25° C.;

extruding the mixture at 70-110° C. in order to obtain small extrudedcylinders suitable for use as subcutaneous implants.

This process cannot be carried out using industrial methods because itis not possible to sufficiently eliminate water from the mixture. Theresults declared in WO 98/109613 cannot therefore be reproduced.

However the fundamental characteristic of the compositions forsubcutaneous implants in the patents referred to above consists of thehomogeneous distribution of the peptide in the polymeric substance,resulting from using a solution of at least one of the two components.

The implants currently on the market have the disadvantage of releasingthe peptides over a limited period of time, generally of around 3months.

SUMMARY OF THE INVENTION

The applicant has now found compositions suitable for preparingsubcutaneous implants which allow the active substance to be releasedover a period of time of at least 6 months.

These compositions consist of a polylactic-glycolic acid (PLGA) and apeptide and have the characteristic of distributing peptide particles inthe PLGA whose dimensions, under microscopic examination, are extremelyheterogeneous, with peptide particles of diameter of between 1 and 60micrometres dispersed in the polymer matrix.

These and other characteristics of the compositions in accordance withthe invention and the process for preparing them will be illustrated ingreater depth in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a microscope test of a cross section of an implantaccording to the invention.

FIG. 2 represents a microscope test of a cross section of an implantaccording to the prior art.

FIG. 3 represents the cumulative amount of avoreline released fromimplants of Example 1.

FIG. 4 represents the cumulative amount of avoreline released fromimplants of Example 2.

FIG. 5 represents the plasmatic concentration of LH, FSH andtestosterone by clinical experimentation using implants having anavoreline content of 10 mg.

FIG. 6 represents the plasmatic concentration of LH, FSH andtestosterone by clinical experimentation using implants having anavoreline content of 15 mg.

FIG. 7 represents the plasmatic concentration of avoreline andtestosterone by clinical experimentation using implants having anavoreline content of 10 mg.

FIG. 8 represents the plasmatic concentration of avoreline andtestosterone by clinical experimentation using implants having anavoreline content of 15 mg.

DETAILED DESCRIPTION OF THE INVENTION

This invention refers to compositions consisting of polylactic-glycolicacid (PLGA) and a peptide suitable for the preparation of long-releasesubcutaneous implants.

The implants prepared from compositions in accordance with thisinvention consist of a PLGA matrix of a high molecular weightincorporating a peptide in the form of particles having extremelyheterogeneous dimensions.

This structure allows the peptide to be released in three stages, whichare, respectively: pure diffusion, diffusion with swelling of the PLGAand release caused by PLGA degradation.

This allows the total release time to be significantly increased.

When these implants are introduced into an aqueous environment the waterdiffuses through the polymer matrix, reaches the peptide particlesclosest to the surface and then the more internal zones, resulting inthe formation of a porous lattice of the hydrated peptide, through whichthe phenomenon of peptide release via pure diffusion (first stage)occurs.

The implant remains unchanged for approximately 6 weeks and over thisperiod releases approximately 30% of the peptide.

The duration of this pure diffusion stage is essentially determined bythe degree of heterogeneity of the dimensions of the peptide particlesand the speed is essentially determined by the peptide content in thePLGA matrix.

As a result of the wide diversity in the dimensions of the peptidegranules, a sufficient quantity of peptide remains after the first stageof dissolution to be released over the subsequent stages.

In the second stage the peptide is released by diffusion with swellingof the polymer.

In the third stage, the residual peptide is released when the matrix isdestroyed.

The succession of the three stages for releasing the peptide withoutdead time depends on the appropriate choice of constituents in thecomposition, in particular:

the heterogeneous nature of the dimensions of the peptide particlesdetermines the first stage of release;

the characteristics of the PLGA (molecular weight and molar ratio) havean influence on the stages of the swelling and matrix degradation.

The technical difficulty of this invention lies in retaining theheterogeneous nature of the dimensions of the peptide particlesthroughout the process for preparing the implants.

This difficulty cannot be overcome by using the known techniques ofmixing by dissolving in a solvent shared by the two compounds or in onesolvent for one of the two compounds, nor by means of the techniques formixing in the melted state.

This difficulty has been resolved in this invention by means of wetgranulation of the PLGA with the peptide. This operation and thesubsequent operations allow the initial heterogeneity of the peptideparticles to be retained.

The invention also refers to the process for preparing thesecompositions and to the aforementioned subcutaneous implants.

One such process is a wet granulation process.

This process consists of the following stages:

a) the peptide in the form of particles having a diameter of between 1and 60 micrometres is homogeneously mixed when dry with PLGA in the formof particles whose granulometry is between 10 to 150, preferably 50 and150, micrometres;

b) the mixture obtained from stage a) is granulated using wetgranulation by adding a suitable liquid, for example ethanol or water;

c) the granulate is then dried until the residual liquid content is0.1-3.0%, preferably 0.5-2.0% by weight. This liquid content isfundamental in that it gives the granule sufficient cohesion to preventthe constituents of the mixture from separating during subsequenttreatments;

d) the mixture obtained from stage c) is extruded. Exposure time in theextruder is from between 1 and 10, preferably between 4 and 6 minutes,with a temperature profile which ranges from 20° C., preferably 30° C.,on entering the extruder to no higher than 120° C., preferably 110° C.,on leaving the extruder. Under these conditions the PLGA melts forming acontinuous matrix which coats the peptide particles while maintainingthe heterogeneous nature of these particle dimensions.

e) the cylinders produced from extrusion may be slightly stretched andthen cut to obtain dimensions suitable for the subcutaneous implants,namely a diameter of between 1.0 and 1.7, preferably between 1.3 and 1.5mm, and a length of between 10 and 30, preferably between 15 and 24 mm;

f) finally, if needed, the cylinders obtained from stage e) aresterilised.

Suitable polylactic-glycolic acid copolymers for use in the inventionhave molecular weights ranging from 50,000 to 150,000 and a molar ratiobetween the lactic acid and the glycolic monomer comprised between 50:50and 95:5.

Preferred polylactic-glycolic acid (PLGA) to be used in the process inaccordance with this invention has a high nominal molecular weight, ofbetween 100,000 and 150,000 D, and a molar ratio between lactic monomerand glycolic monomer of between 70/30 and 75/25.

The peptides which can be used in this invention are preferably, but notexclusively, analogues of LHRH and comprise, for example avoreline:5-oxo-L-prolyl-L-histidil-L-tryptophil-L-seryl-L-tyrosil-2-methyl-D-tryptophil-L-leucyl-L-arginyl-N-ethyl-prolylamide;tryptoreline:5-oxo-L-prolyl-L-histidyl-L-tryptophil-L-seryl-L-tyrosil-D-tryptophil-L-leucyl-L-arginyl-L-prolyl-glicinamide;leuproreline:5-oxo-L-profil-L-histidyl-L-tryptophil-L-seryl-L-tyrosil-D-1eucyl-L-leucyl-L-arginyl-N-etil-L-prolylamide;gosereline:5-oxo-L-prolyl-L-histidyl-L-tryptophil-L-seryl-L-tyrosil-tert-butyl-D-seryl-L-leucyl-L-arginyl-L-prolyl-NH—NH—CO—NH₂.

The peptide preferable used in this invention is avoreline.

The content of peptide in the composition is between 20 and 40%,preferably between 20 and 36% by weight.

The quantity of liquid added for granulation is between 10 and 60,preferably between 20 and 45 parts by weight in comparison with themixture.

The desiccation involved in stage c) takes place at a temperature ofbetween 20-50, preferably between 20-30° C. in a current of dry air.

The transversal section of the cylinders obtained from stage f) revealsunder microscope a heterogeneous structure containing peptide particleshaving the same granulometry as the initial peptide immersed in thematrix constituted by the PLGA.

The cylinders obtained from stage f) can be successfully used forsubcutaneous implants.

Each implant having a diameter of between 1.0 and 1.7, preferablybetween 1.3 and 1.5 mm, and a length of between 10 and 30, preferablybetween 15 and 24 mm, has a peptide content of between 5 and 20 mg.

The peptide released during in vitro and in vivo trials takes place overa time period of at least 6 months.

However the overall time for releasing the peptide can be controlled byvarying the diameter and the length of the implant.

Clinical trials conducted using subcutaneous implants in accordance withthis invention in patients with prostate tumours have shown thetestosterone suppression within 4 weeks with this effect lasting fromapproximately 7 months to approximately 12 months.

In order to illustrate the invention the following Examples are quoted.

EXAMPLE 1

10 grams of avoreline were mixed thoroughly with 30 grams ofpolylactic-glycolic acid (PLGA).

The avoreline had the following characteristics:

acid-alkalimetric titer: 88.1 % by weight;

pKa: 6.15-9.70-12.02;

granulometric distribution : between 1 and 60 micrometres.

The PLGA had the following characteristics:

molecular weight of 116,500 D;

molar ratio between lactic monomer and glycol monomer: 70:30;

intrinsic viscosity (CHCl₃); 0.98 dl/g measured at 25° C.,

granulometric distribution: in 90% of cases between 50 and 150micrometres.

The mixture obtained was granulated wet by adding 16 ml of ethanol usinga 16 mm grid.

The granulate obtained was desiccated for 12 hours at a temperature of25° C. in a current of dry air.

After drying the ethanol content in the granulate was 0.66% by weight.

Finally the granulate was extruded using an extruder with a die head ofdiameter 1.5 mm and length 17.55 mm.

The speed of rotation of the screws was 5 rev/minute and the temperaturewas 30° C. on entering the extruder and 100° C. on leaving the extruder.

The cylinder obtained by the extrusion was slightly stretched and thencut into segments of length 18 mm which were finally radiosterilised. Inthis way implants for subcutaneous use with diameter of 1.5 mm, length18 mm and avoreline content of 25.3% by weight were obtained.

The transversal section of these implants examined under microscopeunder ×275 enlargment reveals a heterogeneous distribution of theparticles of avoreline in the mass of PLGA, as shown in FIG. 1. Inparticular the particles of avoreline retain their initial granulometryof between 1 micrometre ad 60 micrometre.

The same microscopic examination was carried out on an implant producedusing a known technique obtained in accordance with U.S. Pat. No.5,366,734 which under 1100× enlargement reveals a homogeneousdistribution of the two components as in FIG. 2.

Kinetics of in Vitro Release

The implants prepared in accordance with Example 1 were tested in vitroin order to examine the kinetics of releasing avoreline.

The test was carried out under the following conditions.

Five implants were introduced into one flask and the 5 ml of phosphatebuffer at pH 7.4 were added. The test was conducted at 37° C. for aperiod of 210 days, continuously stirring the solution using 100 turnsper minute.

Each week the buffer solution containing the active constituent releasedover the same period was sampled and analysed, while 5 ml of phosphatebuffer were added to the flask as above.

The content of avoreline in the solutions was determined by means ofHPLC under the following conditions:

Column: Vydac 218TP 54300A medium, 5 μm, dimensions 250 x 4.6 mm Mobilephase: 750 ml phosphoric acid 0.1M were added to 250 ml of acetonitrileand the pH was corrected to 2.5 with triethylamine and the mixturefiltered over an FH type filter (millipore). Output: 1.5 ml/minute.Temperature: 30° C. Determination: UV at 220 nm Injection: volume 10 μlAnalysis time: 15 minutes

The results are shown in FIG. 3 in which in the X-axis shows the timeexpressed in days and the Y-axis shows the cumulative quantity ofavoreline released expressed in mg.

EXAMPLE 2

Example 1 was repeated, with the difference that implants were producedwith a diameter of 1.5 mm, length 15 mm and avoreline content of 20.9%by weight.

Microscopic examination produced similar results to Example 1. Theresults of the in vitro release test are shown in FIG. 4 in which theparameters are the same as those in FIG. 3.

EXAMPLE 3

Example 1 was repeated with the difference that PLGA having a molecularweight of 121,900 D was used and implants were prepared having adiameter of 1.5 mm, length 18 mm and avoreline content of 27.9% byweight.

Microscopic examination and the release test produced similar results toExample 1.

EXAMPLE 4

9 grams of avoreline were mixed thoroughly with 24 g ofpolylactic-glycolic acid (PLGA).

The avoreline had the following characteristics:

acid-alkalimetric titer: 90.1 % by weight;

pKa : 6.15 -9.70-12.02:

granulometric distribution: between 1 and 60 micrometres.

The PLGA had the following characteristics:

molecular weight: 121,900 D;

molar ratio between lactic monomer and glycolic monomer: 70:30;

granulometric distribution: in 90% of cases between 50 and 150micrometres.

The mixture obtained was granulated wet by adding 6 ml of water using a1.6 mm grid.

The granulate obtained was desiccated for 12 hours at a temperature of25 ° C. in a current of dry air.

After drying the water content in the granulate was 1.8% by weight.

Extrusion and the subsequent operations were conducted as in Example 1.

The implants obtained had an avoreline titer of 27.6% by weight.

Microscopic examination and the release test produced similar results toExample 1.

EXAMPLE 5

6 grams of tryptoreline were thoroughly mixed with 14 grams of PLGA.

The tryptoreline had a titer of 90.5% by weight and a granulometricdistribution of between 1 and 60 micrometres.

The PLGA had a molecular weight of 121,900 D, a molar ratio betweenlactic monomer and glycolic monomer of 70:30 and a 90% granulometricdistribution of between 50 and 150 micrometres.

The mixture obtained was granulated wet by adding 8 ml of ethanol usinga 1.6 mm grid.

The granulate obtained was desiccated for 12 hours at a temperature of25° C. in a current of dry air.

After desiccation the ethanol content in the granulate was 1.0% byweight.

Extrusion and the subsequent operations were conducted as in Example 1.

The implants obtained had an avoreline titer of 24.7% by weight.Microscopic examination and the release test produced similar results toExample 1.

EXAMPLE 6

6 grams of gosereline were thoroughly mixed with 14 grams of PLGA.

The gosereline had a titer of 89.5% by weight and a granulometricdistribution of between 1 and 60 micrometres.

The PLGA had a molecular weight of 121,900 D, a molar ratio betweenlactic monomer and glycolic monomer of 70:30 and a 90% granulometricdistribution between 50 and 150 micrometres.

The mixture obtained was granulated wet by adding 8 ml of ethanol usinga 1.6 mm grid.

The granulate was desiccated for 12 hours at a temperature of 25° C. ina current of dry air.

After desiccation the ethanol content in the granulate was 1.1% byweight.

Extrusion and the subsequent operations was-carried-out as in Example 1.

The implants obtained had a gosereline titer of 24.9% by weight.

Microscopic examination and the release test produced similar results toExample 1.

Clinical experimentation

Clinical trials were conducted in 60 patients suffering from prostatetumours, using subcutaneous implants prepared in accordance with thisinvention.

A group of patients was treated with implants having an avorelinecontent of 10 mg and a second group was treated with implants having anavoreline content of 15 mg.

The results of the experiments are shown in FIGS. 5 to 8.

The graphs for these Figures can be interpreted as follows:

Graph a) represents the average plasma concentration of the FSH;

Graph b) represents the average plasma concentration of testosterone;

Graph c) represents the average plasma concentration of LH;

Graph d) represents the average plasma concentration of avoreline;

line e) is the line of castration with reference to testosterone.

FIG. 5 and FIG. 7 refer to the use of implants having an avorelinecontent of 10 mg while FIGS. 6 and 8 refer to the use of implants havingan avoreline content of 15 mg.

With reference to FIGS. 5 and 6, the left ordinate shows the plasmaconcentration of LH and FSH expressed in IU/L and the right ordinateshows the plasma concentration of testosterone expressed in nmol/L.

With reference to FIGS. 7 and 8, the left ordinate shows the plasmaconcentration of avoreline expressed in pg/mL and the right ordinateshows the plasma concentration of testosterone expressed in nmol/L.

In all the Figures, the time from implant insertion, expressed in weeks,is shown on the X-axis.

As can be seen from these Figures, using the implants in accordance withthis invention, testosterone is suppressed within four weeks after theimplant is inserted with this effect lasting for a period of betweenapproximately seven months and approximately twelve months.

The plasma concentrations which can be determined for avoreline weremeasured for approximately 6 months after inserting the implant.

What is claimed is:
 1. A composition suitable for the preparation ofsubcutaneous implants having a release time of at least six (6) monthscomprising a polylactic-glycolic acid copolymer (PLGA) and a peptide,said peptide being dispersed in the PLGA matrix in the form of particleshaving heterogeneous dimensions with diameters ranging from 1 to 60micrometers.
 2. The composition as claimed in claim 1, wherein when thepeptide is brought into contact with an aqueous physiological solutionit is released in three stages, with the first stage of releaseinvolving diffusion, the second stage of release involving swelling ofthe PLGA and the third stage of release involving PLGA degradation. 3.The composition as claimed in claim 1, wherein said PLGA has a molecularweight between 50,000 and 150,000 and a molar ratio of lactic monomer toglycolic monomer between 50:50 and 95:5.
 4. The composition as claimedin claim 3, wherein said PLGA has a molecular weight of between 100,000and 150,000 and a molar ratio of lactic monomer to glycolic monomerbetween 70/30 and 75/25.
 5. The composition as claimed in claim 1,wherein said peptide is selected from the group consisting of avoreline,tryptoreline, leuproreline and gosereline.
 6. The composition as claimedin claim 1, wherein said peptide is avoreline.
 7. The composition asclaimed in claim 1, wherein said peptide is tryptoreline.
 8. Thecomposition as claimed in claim 1, wherein said peptide is gosereline.9. The composition as claimed in claim 1, wherein the peptide content isbetween 20 and 40%.
 10. Process for preparing compositions suitable forpreparing subcutaneous implants as claimed in claim 1, comprising thesteps of: a) homogeneously mixing a peptide in particle form whosediameters range from 1 to 60 micrometers, when dry, with PLGA; b)granulating the mixture obtained from step a) by adding a liquid; c)drying the granulate of step b) until the residual liquid content rangesfrom 0.5 to 2.0%, by weight; and d) extruding the mixture obtained fromstep c).
 11. The process as claimed in claim 10, wherein the residencetime in the extruder of step d) is from 4 to 6 minutes at a temperaturewhich ranges from 30° C. on entering the extruder to no higher than 110°C. on leaving the extruder.
 12. The process as claimed in claim 10,wherein the quantity of peptide used in step a) is between 20 and 40%,by weight.
 13. The process according to claim 12, wherein the quantityof peptide is between 20 and 36%, by weight.
 14. The process as claimedin claim 10, wherein when said granulating liquid is ethanol or water,the quantity of liquid added is between 10 and 60 parts per 100 parts,by weight, of the mixture.
 15. Process as claimed in claim 14, whereinsaid ethanol or water is between 20 and 45 parts per 100 parts ofmixture, by weight.
 16. Process as claimed in claim 10, wherein theextrudates obtained by extrusion from step d) have a diameter of between1.0 and 1.7 mm, and a length of between 10 and 30 mm.
 17. Process asclaimed in claim 10, wherein said extrudates have a diameter of between1.3 and 1.5 mm, and a length of between 15 and 24 mm.
 18. Subcutaneousimplants obtained from the composition as claimed in claim
 1. 19.Subcutaneous implants prepared according to the process of claim
 10. 20.Subcutaneous implants as claimed in claim 18, having a diameter ofbetween 1.3 and 1.5 mm, a length of between 15 and 24 mm and a peptidecontent of between 5 and 20 mg.
 21. Subcutaneous implants according toclaim 18, wherein said peptide is selected from the group consisting ofavoreline, tryptoreline, leuproreline and gosereline.
 22. Thecomposition as claimed in claim 1, wherein the peptide content isbetween 20% and 36%, by weight.